Azoarcus olearius BH72 is an endophyte capable of biological nitrogen fixation (BNF) and of supplying nitrogen to its host plant. Our previous microarray approach provided insights into the transcriptome of strain BH72 under N2-fixation in comparison to ammonium-grown conditions, which already indicated the induction of genes not related to the BNF process. Due to the known limitations of the technique, we might have missed additional differentially expressed genes (DEGs). Thus, we used directional RNA-Seq to better comprehend the transcriptional landscape under these growth conditions. RNA-Seq detected almost 24% of the annotated genes to be regulated, twice the amount identified by microarray. In addition to confirming entire regulated operons containing known DEGs, the new approach detected the induction of genes involved in carbon metabolism and flagellar and twitching motility. This may support N2-fixation by increasing energy production and by finding suitable microaerobic niches. On the other hand, energy expenditures were reduced by suppressing translation and vitamin biosynthesis. Nonetheless, strain BH72 does not appear to be content with N2-fixation but is primed for alternative economic N-sources, such as nitrate, urea or amino acids; a strong gene induction of machineries for their uptake and assimilation was detected. RNA-Seq has thus provided a better understanding of a lifestyle under limiting nitrogen sources by elucidating hitherto unknown regulated processes. Only certain prokaryotes have the capability to convert atmospheric N2 into combined forms for plant assimilation. This process is termed biological nitrogen fixation (BNF), and the prokaryotes that perform BNF are referred to as diazotrophs. Harnessing the power of BNF grants us a better alternative for chemical fertilizers, thereby taking us a step closer to sustainable agriculture [Azoarcus olearius BH72, is a diazotroph belonging to the class Betaproteobacteria [Leptochloa fusca L. Kunth) from the Punjab region of Pakistan [A. olearius while fixing nitrogen.Nitrogen, a component of biomolecules such as proteins and nucleic acids, is important for the growth of plants . In spitbacteria . It is aPakistan . EarlierPakistan . This stPakistan ,7. AnothPakistan . This stPakistan . Hence, 2-fixing and ammonium-grown conditions [A. olearius BH72 [nifH, fdxN, draG, amtB, and amtE [Azoarcus provides the necessary energy for BNF. A recent study has revealed that in E. coli that has been artificially engineered to perform BNF, the metabolic flux shifted from the tricarboxylic acid (TCA) cycle to the pentose phosphate pathway (PPP) and to ethanol fermentation to support BNF [As an initial step in this quest, we previously used microarrays to compare the transcriptome of strain BH72 under Nnditions . For thiius BH72 . As expeius BH72 , and espius BH72 . Therefoius BH72 ,13. Neveand amtE ,15,16,17port BNF . In straAzotobacter vinelandii, almost 30% of the annotated genes were found to be differentially expressed during N2-fixing growth in comparison to non-fixing growth [Paenibacillus sp. WLY78; roughly 60% of the genes were detected to be regulated [RNA-Seq overcomes many of the above limitations of microarrays. RNA-Seq has a higher sensitivity, specificity, and increased dynamic range of detection. Additionally, studies have already shown that RNA-Seq approaches reveal information and regulatory features in addition to the data from microarray analysis ,22,23,24g growth . The quaegulated . These aAzoarcus olearius strain BH72 was initially grown in minimal medium  [2-fixing conditions, strain BH72 was cultivated microaerobically (0.3% O2) in a bioreactor  at 37 °C with constant stirring at 600 rpm in 1.6 L of N-free SM medium in the presence of N2. As a control, the strain was also grown in ammonium-based conditions by supplementing the SM medium with 10 mM NH4Cl. Under both conditions, three independent cultures were set up. An amount of 30 mL of the cultures was harvested at an optical density (OD578) of 0.5–0.7 by centrifuging at 5500× g and 25 °C for 10 min. The supernatant was removed, and the pellets were frozen in liquid nitrogen and were stored at −80 °C.ate, SM)  without v/v/v)) (pH 4.0)  and NAES  SDS at pH 5.1) and was heated to 65 °C. The cell suspension was incubated at 65 °C for 5 min, further incubated on ice for 10 min, and then centrifuged at 13,000× g and 12 °C for 15 min. The aqueous phase was extracted again with an equal volume of phenol–chloroform–isoamyl alcohol (pH 4.0), and this step was repeated thrice. The resulting aqueous phase was mixed with an equal volume of a chloroform–isoamyl alcohol mixture (24:1 (v/v))  and was extracted after centrifugation at 6300× g and 12 °C for 5 min. After extraction, the nucleic acids were precipitated with an equal volume of isopropanol , were incubated on ice for 60 min, and were centrifuged at 13,000× g and 4 °C for 10 min. The pellet was washed with the same volume of 70% ethanol  and was centrifugated at 4 °C for 5 min. After drying, the pellet was re-suspended in 30 µL of RNase free water. The sample was immediately frozen in liquid nitrogen and was stored at −80 °C for further processing.RNA extraction was performed by means of the hot phenol method, as described previously . In brieFrom the extracted nucleic acids, genomic DNA was eliminated using the RNeasy Plus Mini Kit  as per the protocol prescribed by the manufacturer for the purification of total RNA containing small RNAs. In the procedure, 10 µg of the extracted nucleic acid was used as the sample instead of cells.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE176473).The RNA samples were further processed and sequenced at Vertis Biotechnologie AG, Freising, Germany. The ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria . The samples were fragmented by four pulses of ultrasound that were 30 s each at 4 °C. To aid in directional sequencing, a 3′ Illumina TruSeq sequencing adapter with barcodes was ligated to the 3′ end of the fragmented RNA molecules. The first-strand cDNA was synthesized using M-MLV reverse transcriptase and a primer binding to the adapter sequence. Synthesized cDNA was purified, and to its 3′ end, the 5′ Illumina TruSeq sequencing adapter, which also contained barcodes, was ligated. The cDNA was amplified through 13 cycles of PCR using a high-fidelity DNA polymerase to obtain about 10–20 ng/µL of DNA. The amplified cDNA was purified using an Agencourt AMPure XP kit . The six samples were pooled in approximately equimolar amounts and were size-fractionated using a preparative agarose gel to obtain a size range between 200 and 400 bp. The sample pool was sequenced on an Illumina NextSeq 500 system using a 75 bp read length. The sequence of the TruSeq adapters and the barcodes used in this procedure are provided in Azoarcus olearius strain BH72 using the READemption tool [The Illumina sequencing reads were trimmed using the fastptool  based onion tool  with itsion tool . In the gapA gene (azo2837) as the internal reference. A list of gene-specific primers and annealing temperatures for each pair are provided in The differential expression of selected targets identified by RNA-Seq was verified through quantitative RT-PCR of RNA samples. For each target, the first-strand cDNA was synthesized by Superscript III reverse transcriptase . Specifically, 2.5 µM of gene-specific reverse primer and 20 ng of total RNA were used in a 10 µL reaction volume and were incubated at 55 °C. Other components and steps were followed as per the prescribed protocol. In each reaction, 10 U of Recombinant RNasin Ribonuclease Inhibitor  was added instead of RNaseOUT. For quantification, 1 µL from the reverse transcription reaction was further amplified using SsoAdvanced Universal SYBR Green Supermix  with 0.3 µM of both forward and reverse primers in a 20 µL reaction volume. The reaction was initiated with an initial denaturation (98 °C for 2 min) followed by 40 cycles of two-step amplification  in a CFX96 Real-time PCR Detection System . The melting curve of the product was analyzed by heating the samples from 65 °C to 95 °C in 0.5 °C increments with a 5 s incubation period at each step. The PCR efficiency was verified using the LinRegPCR tool . RelativAzoarcus olearius BH72 grown microaerobically under N2-fixing and non-fixing conditions (with ammonium chloride as nitrogen source). Cells and RNA were obtained from three independent experiments for each condition. The RNA-Seq approach yielded more than 10 million reads for each processed sample. Out of these, 83–88% of the reads aligned to the genome of Azoarcus , respectively, were detected to be transcribed with a minimum reads per Kilobase per Million mapped reads (RPKM) value of 10 in all three biological replicates. Together, this constituted 3300 unique ORFs (82% of the 3989 annotated ORFs). As expected, the Euclidean distance between the samples, using the count data after variance-stabilized transformation, indicated a closer relationship between the biological replicates grown in the same conditions compared to the distance between the replicates from different growth conditions  less than 0.05  under N2-fixing conditions [Paenibacillus sp. WLY78, an astounding 60% of the annotated genes were reported to be differentially regulated  under N2-fixing conditions [2) grown culture as the control. Hence, a considerable portion of the genes that were detected to be differentially expressed might be functionally important for growth at high versus low oxygen concentrations. In a microarray-based transcriptome study in Azoarcus, we showed that at least 8.7% of the annotated ORFs were differentially expressed during microaerobic growth when compared to aerobic conditions [Using the DESeq2 tool , differehan 0.05 . Out of nditions . In Paennditions . In contnditions .2-fixing and ammonium-grown A. olearius using the microarray approach under otherwise comparable experimental conditions [A. olearius. DEGs uniquely identified by RNA-Seq included genes that encoded for proteins involved in vital pathways such as those for nitrogen assimilation, energy production, motility, and vitamin biosynthesis.We had previously compared the transcriptome of Nnditions . This stAlong with these DEGs, RNA-Seq identified a minor proportion of 44 genes to exhibit an opposite modulation of expression compared to the microarray results . To subs2-fixing conditions. To further comprehend the list of DEGs detected by RNA-Seq, we classified them based on the cluster of orthologous groups (COGs) of the proteins encoded by them , coenzyme metabolism (H), and replication and recombination machinery (L) had a higher frequency of down-regulation. When compared to the microarray approach, in general, RNA-Seq detected a higher frequency of enhanced expression and suppression in most of the COG categories . This included the genes that are responsible for the regulation of nitrogenase genes, FeMo cofactor synthesis, nitrogenase synthesis, and electron transport. Still, microarrays did not detect the regulation of all of the corresponding genes. Enhanced expression of important genes such as nifL, nifH, nifK, nifU, and fdxD were not detected. This shortfall was overcome by the robust and more sensitive RNA-Seq method, which detected most of the genes within this cluster to be differentially regulated  and type 2 as well as one of the two type 6 protein secretion systems (tss-1 or imp) , which demonstrated an 18-fold induction [The  or imp) b. Type 6ius BH72 . This wanduction , similarWe observed a similar trend in the genes that were suppressed, but they were scarce and less pronounced compared to the up-regulated genes c.2. This included assimilatory nitrate reduction, high-affinity ammonium transporters, and urea metabolism. For assimilatory nitrate reduction, A. olearius engaged the ABC-transporter NasFED to transport nitrate into the cytoplasm, where it is reduced to nitrite by NasA and NasC. Further, nitrite is commonly reduced to ammonium by the NADH-dependent nitrite reductase complex NirBD. Genes encoding for all of these proteins were up-regulated by A. olearius. Additionally, genes encoding channels for nitrate/nitrite transport, narK, and nirC were also induced. All of these DEGs, except for narK, were induced by more than 50-fold; some of them reached over 200-fold , glutamate/aspartate transport (glnHPMQ), and three genes of the branched-chain amino acid transport . The article further proposes that the diazotrophs, while consuming the low molecular weight nitrogen sources, might already start fixing N2. N2-fixation is conducted as a buffer until it shifts to the breakdown and assimilation of the abundant high molecular weight sources in the soil environment. They propose that the continuous energy requirement of N2-fixation might be a drawback in comparison to the assimilation of high molecular weight sources, which needs a one-off high investment. This hypothesis is further supported by previous studies describing pathways that exert tight control on N2-fixation and immediately switch-off nitrogenase activity upon detecting a sufficient amount of ammonium [Recently, it was proposed  that in ammonium ,39,40.Azoarcus appears to search for low-cost nitrogen sources. A high, constant energy demand and the requirement to maintain a stable microaerobic environment for biological nitrogen fixation might favor this search. Although this model supports the behavior detected in our pure culture experiment, it would be interesting to explore if and how Azoarcus modifies this regulation once it is in a symbiotic relationship with plants.Even while fixing nitrogen, flhDC genes encoding for the transcriptional regulators responsible for the expression of flagellar motility-related genes. Earlier studies have shown that the flhDC operon in E. coli is regulated by RpoN and that a rpoN knockout mutant had a reduced expression of flagellar related genes with a concurrent reduction in motility [fliC3) was also found to be induced. Strain BH72 contains three paralogs that encode flagellins. The flagellin encoded by fliC3 was found to be the major constituent of the flagellar structure and to be responsible for swarming activity [E. coli, fliC was found to be up-regulated during N2 starvation, and a subsequent CHIP-Seq analysis revealed the binding of NtrC to the promoter sequence of fliC [2-fixation.As discussed earlier, among the COG categories, cell motility has a higher proportion of enhanced expression than suppression. Strain BH72 contains at least 77 genes annotated to be encoding for proteins related to the assembly and function of flagellar motility and chemotaxis . Out of motility ,43. Nextactivity . In E. c of fliC . These epilS, which is located upstream of the pilAB operon, was found to be suppressed by 1.79-fold  and ethanol fermentation. The authors hypothesize that this shift could potentially aid in the rapid response to energy demand [Azoarcus does not encode glucose transporters [2-fixation. Therefore, we looked at other means of carbon assimilation and energy production.Biological nitrogen fixation is an energy-intensive process, as reducing each mole of nitrogen requires at least 16 moles of ATP. Along with fixing Ny demand . Howeversporters  and cannsporters . Hence, 2-fixation within root nodules. Specifically, mutants of S. meliloti in genes encoding for isocitrate dehydrogenase (icd), 2-oxoglutarate dehydrogenase, and citrate synthase (gltA) formed root nodules but did not fix nitrogen [Strain BH72 encodes transporters of dicarboxylic acids  and has nitrogen ,48,49,50sdhC and sdhD) were detected to be down-regulated by 1.7-fold. This enzyme is also involved in quinone reduction for oxidative phosphorylation. Additionally, with the exception of a few isolated genes, we did not detect any clear induction or suppression pattern among the genes encoding for the enzymes involved in oxidative phosphorylation. It is most likely that their expression is restricted due to the limited availability of the electron acceptor, oxygen, under microaerobic conditions. In turn, this limits the generation of ATP. Additionally, not regenerating the reducing equivalents could indirectly affect the flux through the TCA cycle. Studies have shown that bacteria with reduced oxidative metabolism or a high redox ratio (NADH/NAD) increases the metabolic flux through the overflow metabolism [pta (encoding for phosphate acetyltransferase) and ackA (encoding for acetate kinase)  . Interesc growth .2-fixing bacteria utilize the lipid biogenesis pathways that synthesize poly-ß-hydroxybutyrate (PHB), fatty acids, or both [2-fixing conditions [phbC2. This gene encodes for PHB synthase, the enzyme that catalyzes the final step in the three-step synthesis of PHB from acetyl-CoA. Moreover, the induction of two probable phasins  was also detected. In B. japonicum, it was shown that the phasins were more important in PHB accumulation than the metabolic enzymes involved in their synthesis [To reduce the redox ratio, N or both ,55,56. Snditions . In the ynthesis . ConcernThe suppression of two enzymes in the pentose phosphate pathway (PPP) was detected. The genes for transketolase (Tkt) and ribose-phosphate diphosphokinase (PrsA) were 1.9-and 4.4-fold suppressed, respectively. As the products of PPP are substrates for several synthetic pathways, its suppression could have ramifications on downstream metabolisms such as those for purine and riboflavin synthesis (discussed later).Overall, it seems that activities under diazotrophic growth probably demand increased energy production and that strain BH72 responded to the high energy demand by inducing the expression of TCA cycle components. Nevertheless, growth under oxygen-limited conditions restricts the capacity of oxidative phosphorylation, thereby increasing the redox ratio and, in turn, limiting the TCA cycle. Strain BH72 may overcome this hurdle by the induction of overflow metabolism and PHB synthesis.12) synthesis, a cofactor for vital metabolic enzymes. A cobalamin-dependent ribonucleotide reductase is essential for the plant colonization of S. meliloti [A. vinelandii, cobalamin synthesis was more dependent on the concentration of cobalt than on the availability of combined nitrogen [12 activity [Many microorganisms encode the machinery for cobalamin  synthesis were detected to be down-regulated , which was induced by 34-fold, which may act complementarily.Similarly, the genes involved in riboflavin , which is a substrate for purine synthesis. Subsequently, we detected genes involved in the purine biosynthesis to be down-regulated . This wosubtilis ,66. In t2-fixation, which is in accordance with the reduced growth rate of strain BH72 under N2-fixation in comparison to ammonium-grown conditions [We had previously highlighted DEGs encoding for proteins involved in translation (COG category J). In total, 51 genes encoding for proteins related to ribosome biogenesis and functioning were found to be down-regulated . The lisnditions .nifA (80-fold) coding for the transcriptional activator of the nif genes. ntrC, hoxA, and modE were the other well studied induced regulators. They have an established link to nitrogen metabolism or nitrogen fixation. HoxA, a hydrogenase transcriptional regulatory protein, regulates the expression of structural genes for the hydrogenase uptake system in free-living B. japonicum and Rhodopseudomonas palustris [hupS, was also substantiated by RT-qPCR  genes at high molybdate concentrations [modE gene is located in the same cluster as the genes encoding for the transporter (modA1B1C1). Correspondingly, RNA-Seq detected the induction of all of these genes. A similar regulation was also detected for modD, which was located outside of this cluster. Studies in Azotobacter vinelandii, Paenibacillus sp. WLY78, and Rhodobacter capsulatus have shown that the expression of modABC is coordinated with the expression of the Mo-Nitrogenase genes [modA impaired the Mo-Nitrogenase activity of B. japonicum within the root nodules of soybean plants under Mo-limiting conditions [azo1949 and azo2458, that are 20-and 5-fold induced, respectively.RNA-Seq detected 33 DEGs that encode transcriptional factors and response regulators. Out of these, 23 were induced, and 10 were suppressed . For elealustris ,68,69. I RT-qPCR . Hydrogefixation . ModE, ttrations ,72,73. Ise genes ,26,74. Anditions . Strain azo2893 was identified by RNA-Seq to be repressed, while the microarray had indicated an induction. RT-qPCR analysis of the samples used for RNA-Seq substantiated the detected repression . Additionally, in E. coli, both yeaG and yeaH are present within the same cluster. The promoter of yeaG was bound by NtrC under N-starvation [RNA-Seq detected two neighboring genes, arvation . Subsequarvation . The absarvation . The hetazo2278, azo2279, and azo2280) encoding for hypothetical proteins . Azo2278 has two predicted nitroreductase domains, albeit with very low similarity. Azo2280 seems to be unique to the Azoarcus genus, while the first two genes were widespread among prokaryotes. Considering the variety of functions performed by proteins containing nitroreductase domains [2-fixation is compelling.Strong induction was detected for the cluster of three genes (2-fixation lifestyle through inducing carbon metabolism and motility and by suppressing translation and vitamin biosynthesis (2 as a N-source. Simultaneously, it has ignited the quest to explore the role of non-coding RNA under N2-fixation and the regulation of N-assimilation in an endophytic lifestyle. The directional RNA-Seq used in this study revealed the transcription of several antisense and small RNAs. This has been recently reported in several prokaryotes as well [2-fixation. This new and vast field necessitates a separate study on its own.With the new data from the reported RNA-Seq analysis, strain BH72 appears to support a Nynthesis . Still, ynthesis . Thus, R as well ,80,81. I


Several studies have explored the role of the inflammatory cells and cytokines involved in the protection or pathogenesis of coronavirus disease 2019. Unfortunately, the results have been controversial, and further studies are needed to better understand not only the roles but also the balance of these parameters, which are crucial data to improve prevention and treatment. As COVID-19 has a well-determined phasic progression and rapidly deteriorates approximately seven days after the onset of symptoms, it is extremely necessary to detect the clinical signs that are predictive of the outcome as early as possible. To this end, in this preliminary study, we evaluated the data relating to the monocyte intracellular TNF-α expression and lymphocyte subpopulations in peripheral blood collected from patients at admission and every day of hospitalization until day 7. Our findings point to a modulation of the different cellular mediators of the immune system, which probably play a key role in the outcome of the coronavirus disease 2019.In December 2019, a novel coronavirus, “SARS-CoV-2”, was recognized as the cause of coronavirus disease 2019 (COVID-19). Several studies have explored the changes and the role of inflammatory cells and cytokines in the immunopathogenesis of the disease, but until today, the results have been controversial. Based on these premises, we conducted a retrospective assessment of monocyte intracellular TNF-α expression (iTNF-α) and on the frequencies of lymphocyte sub-populations in twenty-five patients with moderate/severe COVID-19. We found lymphopenia in all COVID-19 infected subjects compared to healthy subjects. On initial observation, in patients with favorable outcomes, we detected a high absolute eosinophil count and a high CD4+/CD8+ T lymphocytes ratio, while in the Exitus Group, we observed high neutrophil and CD8+ T lymphocyte counts. During infection, in patients with favorable outcomes, we observed a rise in the lymphocyte count, in the monocyte and in Treg lymphocyte counts, and in the CD4+ and in CD8+ T lymphocytes count but a reduction in the CD4+/CD8+ T lymphocyte ratio. Instead, in the Exitus Group, we observed a reduction in the Treg lymphocyte counts and a decrease in iTNF-α expression. Our preliminary findings point to a modulation of the different cellular mediators of the immune system, which probably play a key role in the outcomes of COVID-19. In late December 2019 in Wuhan, Hubei Province, China, several patients with severe pneumonia of unknown origin were reported from different hospitals . It is aA total of twenty-five patients hospitalized between 4 March 2020 and 12 April 2020 for respiratory insufficiency at the ‘Azienda Ospedaliera dei Colli Monaldi-Cotugno Hospital’, Italy, diagnosed with SARS-COV-2 infection and who were negative for common respiratory pathogens were considered for this study. All patients were hospitalized according to their respiratory insufficiency and without a previous COVID-19 test. Upon admission to the hospital, they were tested for COVID-19, and they their test results showed that they were positive for COVID-19 infection . As indicated by WHO guidelines, confirmation of SARS-CoV-2 infection was obtained through RT-PCR positivity via an oropharyngeal swab. Following manufacturer’s instructions on a MicrobScan real-time PCR , we used the Allplex™ SARS-CoV-2 multiplex real-time PCR assay  to detect 4 target genes: RdRP, S, and N genes specific for SARS-CoV-2 and the E gene, which is expressed in all Sarbecovirus’, including SARS-CoV-2. A positive result  for all viral targets indicated the presence of SARS-CoV-2 RNA in the sample. Data including demographic, medical history, symptoms, signs, and laboratory findings including blood routine, lymphocyte subsets, and infection-related biomarkers were collected from patients’ medical records. The patients were classified on the basis of their outcome: the Exitus Group, which was composed of patients who never recovered from COVID-19 and who died during infection, and the Good Prognosis Group, which was composed of patients who recovered from COVID-19. The intracellular TNF-α and lymphocyte subpopulations were evaluated in peripheral blood collected from patients on alternate days. Whole blood samples were collected at admission (none of the patients received any treatment before blood sampling) and every day of hospitalization until day 7  in tubes containing EDTA or sodium-heparin, which were then immediately analyzed using flow cytometry. Blood counts were quantified as per institutional protocols using a Sysmex XN-3000 instrument . The study was conducted according to the guidelines of the Declaration of Helsinki and was approved on 8 July 2020 by the Ethical Committee of the ‘AORN Ospedali dei Colli—Monaldi—Cotugno—CTO, Napoli, Italy’, approval number AOC-0020053-2020. Informed consent was obtained from all of enrolled patients for the use of their biological samples and clinical data for the purposes of clinical research and the study of diseases.Data were acquired using a Navios 10C\3L Flow Cytometry Instrument  and data analysis was performed using Kaluza software  following the manufacturer’s instructions. Analyses of the T, B, NK, and Treg lymphocyte subpopulations were performed on whole peripheral blood collected in an EDTA vacutainer while analysis of monocytes, TNF-α, and HLA-DR expression were performed on peripheral blood samples collected in sodium-heparin. To evaluate the percentage and the absolute value of the lymphocyte subpopulations, 2 kits were used, both from Beckman Coulter Inc. , containing mixes of monoclonal antibodies bound to different fluorochromes and were following the manufacturer’s instructions: the AQUIOS Tetra-1 Panel kit, consisting of CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5, and the AQUIOS Tetra-2+ Panel kit, consisting of CD45-FITC/(CD56+CD16)-RD1/CD19-ECD/CD3-PC5. The subpopulation of Treg lymphocytes (CD3+ CD4+ CD25+ FoxP3+) was evaluated with the DuraClone IM Treg Tube Kit  following the manufacturer’s instructions. For the evaluation of the monocytes, for the percentages of iTNF-α positive and HLA-DR positive monocytes (% positive cells) among the total monocyte population, the Duraclone IF Monocyte Activation Tube Kit  was used, following the manufacturer’s instructions and according to the methods previously described by Monneret G. et al. . The prop-value < 0.05 confirmed a Gaussian distribution of the data. The difference between the groups was evaluated graphically using box and whiskers plots. The comparison between the groups is reported as mean ± SD. Statistical significance was obtained through paired or unpaired Student’s t-tests. In all the of the analyses, p-values < 0.05 were considered as statistically significant. The best cut-off was derived using the Cutoff Finder software, a freely available web application enabling rapid biomarker cutoff optimization .Data obtained from the analysis were analyzed for statistical significance using the GraphPad 9.0.0 software. Patient baseline characteristics were described using descriptive statistics. Pearson’s test was used to evaluate the distribution and a value of Patient demographics are summarized in p < 0.05), but no differences were observed between the Good Prognosis vs. Exitus groups at the first determination (day 0) (p = 0.684) (p < 0.05), while in Exitus Group, the average remained lower, and no statistically significant differences were observed  (p = 0.469) (p < 0.05) but not in Exitus Group (p = 0.290) (p < 0.05) (p < 0.05) (p > 0.05).At admission, we evaluated ALC, AMC, ANC, AEC, PLT, NLR, PLR and, in addition, we analyzed ALC and AMC during the course of infection, for a total of eight determinations. We observed a statistically significant reduction in ALC compared to healthy subjects  . On the = 0.725) . We also= 0.469) , but fro= 0.290) . For ANC < 0.05) . We foun < 0.05) . No diff < 0.05) . Using t < 0.05)  and PLR  < 0.05)  between p = 0.469) (p < 0.05) (p = 0.684) and between the first and third determinations vs. the fourth to last (p = 0.537) (data not shown). Therefore, we evaluated the absolute count of the CD4+ and CD8+ lymphocytes and CD4+/CD8+ lymphocyte ratio in the two groups. At the first determination, the Exitus Group showed a significantly higher count of CD8+ lymphocytes compared to the Good Prognosis Group  (p = 0.758) (p < 0.05) (p < 0.05) (p < 0.05), while in the Exitus Group, we did not observe statistically significant differences in the CD4+ and CD8+ counts. Moreover, at the first determination, the Good Prognosis Group showed a higher statistically significant CD4+/CD8+ ratio than the Exitus Group  , and no differences were observed for the Exitus Group (p = 0.349) (p < 0.05) in the Good Prognosis Group, while in the Exitus Group, we observed a significant reduction in Treg lymphocytes  (p = 0.651) and among the first and third and after the fourth determination onwards . We also obtained same results for the NK lymphocytes subset, in which no differences were observed between the two groups at the first observation  and among the first and third and after the fourth determination onwards  (data not shown).All data relating to monocyte iTNF-α and HLA-DR expression on and lymphocyte subpopulations frequency were evaluated using peripheral blood collected from patients on alternate days, starting from hospitalization, for a total of eight determinations. At the first determination, the good prognosis patients showed percentages of iTNF-α positive monocytes (% positive cells) that were lower than those of the Exitus Group, but no statistically significant difference was observed  . Further= 0.469) c,d in th= 0.469) c compare= 0.469) d, in whi < 0.05) . In addi < 0.05) , and no = 0.758) . After t < 0.05)  and CD8+ < 0.05)  counts c < 0,05) . Between= 0.349) . After t= 0.349) . However < 0.05) . FinallyThe interaction between COVID-19 and the immune system is complex. Infection correlates to an extensive infiltration of the neutrophils and macrophages into the lungs that leads to pro-inflammatory cytokine production, and, in addition, involves CD8+ and CD4+ T cells that orchestrate the immune response against viruses . UnfortuAlteration in monocyte count, phenotype, and function plays a significant role in the SARS-CoV-2 pathogenesis, as reported in many studies, and depends on the stage of the disease and is variable in different manuscripts ,69. In COverall, the first COVID-19 wave and the related health emergency have found the majority of the hospitals and clinicians to be unprepared around the world. For this reason, the management of patients and the planning of research analyses on an optimal cohort of infected subject was incredibly difficult considering the little information on COVID-19 that was available during first emergency. It needs to be considered that the 25 patients enrolled in this study were hospitalized during the first COVID-19 wave/emergency, Italy was suggesting that subjects with COVID-19 symptoms remain at home for as long as possible so as to avoid congesting the hospitals due to the lack of hospital beds dedicated to COVID-19 patients. For this reason, all of the patients enrolled in this study were hospitalized as a consequence of their respiratory insufficiency and without a previous COVID-19 test. Once they were admitted to the hospital, they were tested for COVID-19 infection, and the test results showed that the participants were positive for COVID-19 infection. As a result, the day of onset corresponds to day 0/the first determination, and for this reason, we did not evaluate the impact of the time from symptom onset to admission to the hospital based on the severity of the illness. Considering these points of view, our preliminary results need to be validated in a larger, homogeneous and well-defined cohort of patients. Additionally, the potential drawbacks of our study remain due to the fact that we only included 25 patients, and we did not include a cohort of healthy patients on which we could have conducted the same analyses. We also did not provide a comparison with soluble TNF-α or others soluble mediators in whole blood.Many studies have revealed that monocytes and macrophages can induce pathogenesis during the COVID-19 through dysregulated function, including the enhancement of inflammatory cytokines and chemokines. For these reasons, discovering the involved mechanisms and mediators can be significantly helpful to develop novel therapeutic methods and cell-based drugs for alleviating SARS-CoV-2 infection. Considering that COVID-19 disease has a well-determined phasic progression and rapidly deteriorates approximately seven days after the onset of symptoms, it is crucial to identify predictive clinical biomarkers as early as possible to better assist patients. In this vision, we have done a preliminary longitudinal analysis on COVID-19 infected patients, evaluating the rise and fall of different mediators of the immune system during infection. The preliminary data collected in our study point to a modulation of the different cellular and soluble mediators of the immune system during infection, which probably play a key role in the outcome of COVID-19. Based on our preliminary findings, further studies are needed to better understand the roles and balance of the different immune cell subsets involved in the protection or pathogenesis of COVID-19 in order to detect the clinical signs that are predictive of outcome as early as possible.


Energy-dependent mechanisms favoured HCE-2 transfection, whereas protein production was influenced by energy-independent uptake mechanisms. Nanovectors with a mean particle size between 94 and 348 nm and a positive superficial charge were formulated as eye drops containing 1% (w/v) of polyvinyl alcohol (PVA) with 7.1–7.5 pH. After three days of topical administration to mice, all formulations produced GFP in the corneal epithelium of mice. SLNs allowed the obtaining of a higher transfection efficiency than naked mRNA. All formulations produce IL-10, and the interleukin was even observed in the deeper layers of the epithelium of mice depending on the formulation. This work shows the potential application of mRNA-SLN-based nanosystems to address corneal inflammation by gene augmentation therapy.The anti-inflammatory cytokine Interleukin-10 (IL-10) is considered an efficient treatment for corneal inflammation, in spite of its short half-life and poor eye bioavailability. In the present work, mRNA-based nanomedicinal products based on solid lipid nanoparticles (SLNs) were developed in order to produce IL-10 to treat corneal inflammation. mRNA encoding green fluorescent protein (GFP) or human IL-10 was complexed with different SLNs and ligands. After, physicochemical characterization, transfection efficacy, intracellular disposition, cellular uptake and IL-10 expression of the nanosystems were evaluated  The eye is considered the perfect target for gene therapy due to its physiological features: it is easy to access and examine, it has a well-defined anatomy and it is relatively immune privileged. Moreover, from an experimental point of view, in the same subject, one eye can be used as a control, whereas the other eye can be used as the experimental target ,2,3. Curin vitro and in vivo gene expression. Particularly, mRNA therapeutics present several advantages: since mRNA does not need to enter inside the cell nucleus in order to exert its effect, protein translation is faster than in the case of DNA; furthermore, mRNA expression is temporary and it owns a safe profile with no risk of insertion-related mutagenesis, as it does not incorporate into the genome of the host [In the last few decades, a wide range of reagents and techniques suitable to transfer nucleic acids into cells have been developed, in order to modulate both the host . Additiothe host , which mthe host . In thisthe host . Presentthe host ,13,14. Ithe host ,16. Thesthe host ,18.Among lipid-based systems, liposomes and solid lipid nanoparticles (SLNs) have been suggested as up-and-coming non-viral ocular drug delivery systems. ,20. Lipode novo production of IL-10 in corneal cells. Additionally, vectors prepared with plasmid DNA (pDNA) were evaluated for comparison purposes. Formulations containing mRNA or pDNA encoding green fluorescent protein (GFP) or IL-10 were characterized from a physico-chemical point of view, and then evaluated for internalization and transfection properties, both in vitro by using human corneal epithelial (HCE-2), and in vivo after administration to mice by ocular instillation.The aim of this experimental work is the development of SLNs containing mRNA as non-viral vectors, to be used as a genetic supplementation strategy for the ® ATO 5  was kindly provided by Gattefossé . Tween 80 and dichloromethane were purchased from Panreac  and sodium behenate from Nu-Chek Prep . Sigma-Aldrich  provided protamine sulfate salt from salmon (Grade X) (P), dextran (Mn of 3260 Da) (DX), DEAE-dextran, Nile Red and partially hydrolyzed polyvinyl alcohol (PVA) 9000–10,000 Da Mw. Lifecore Biomedical  supplied hyaluronic acid (HA) (Mw of 100 KDa).1,2-Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) was obtained from Avanti Polar-lipids, Inc. . PrecirolTM EGFP mRNA (5moU) and CleanCapTM Cyanine 5 EGFP mRNA . Both of the nucleic acids encode green fluorescent protein (GFP). mRNA encoding human IL-10 was customized by TriLink BioTechnologies. Professor BHF Weber laboratory  kindly provided plasmid pcDNA3-EGFP (6.1 kb) encoding GFP. Plasmid pUNO1-hIL10 (3.7 kb), which encodes human IL-10, was acquired by InvivoGen . Label IT® Cy®5 Nucleic Acid Labeling Kit was obtained from Mirus Bio .CleanCapTM RNase I was purchased from Life Technologies , Deoxyribonuclease I (DNase I) and sodium dodecyl sulfate (SDS) from Sigma-Aldrich and GelRedTM from Biotium .Materials used on agarose gel in an electrophoresis assay were acquired from Bio-Rad . AmbionTM (DMEM/F-12 with GlutaMAXTM), fetal bovine serum (FBS), Penicillin-Streptomycin, Trypsin/EDTA and Attachment Factor were acquired from Life Technologies , whereas epidermal Growth Factor (EGF) and human insulin solution were obtained from Myltenyi Biotec  and Sigma-Aldrich , respectively. The DuoSet Ancillary reagent kit and Enzyme-linked immunoassay (ELISA) for IL-10 were obtained from R&D Systems .Human Corneal Epithelial (HCE-2) cells were purchased from American Type Culture Collection . Cell culture reagents, including Dulbecco’s Modified Eagle´s Medium/Nutrient Mixture F-12 with GlutaMAXTM 2000 Lipid-Reagent was acquired from Life Technologies . The 7-Amino-Actinomycin D (7-AAD) Viability Dye was acquired from Beckman Coulter .Reporter lysis buffer was provided by Promega Biotech Ibérica . Paraformaldehyde (PFA) was obtained from Panreac, while 40,6-diamidine-20-phenylindole dihydrochloride (DAPI)-fluoromount-G was purchased from Southern Biotech . Phosphate buffered saline (PBS) and HEPES buffered solution were obtained from Gibco  and Lipofectamine® O.C.T.-compound.Antibodies used for immunoassay, Anti-GFP polyclonal antibody and goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488, were provided by Life Technologies , except rabbit Anti-IL-10 antibody, which was obtained from Abcam . Sakura Finetek Europe  supplied Tissue-TekOther chemicals, unless detailed, were reagent grade from Panreac  and Sigma-Aldrich .EE), hot-melt emulsification (SLNHM) and coacervation (SLNC).Three different techniques were used in order to prepare SLNs: solvent evaporation/emulsification  of the organic phase (Precirol® ATO 5 dissolved in dichloromethane 5% w/v) in the aqueous phase, containing the cationic lipid DOTAP (0.4% w/v) and Tween 80 (0.1% w/v). SLNs’ precipitation occurred upon dichloromethane evaporation.SLNreported , with thHM was produced as a SLNEE with minor modifications. Briefly, DOTAP (0.4% w/v) and Tween 80 (0.1% w/v) were dissolved in water. This aqueous solution and Precirol® ATO 5 were heated in a bain-marie. When both phases were led to 80 °C and lipids melted, the aqueous solution was added to the Precirol® ATO 5. The emulsion was obtained by sonication  for 30 min at 50 W. SLNs were obtained by cooling the obtained nanoemulsion on ice for 30 min. SLNC were composed of behenic acid as the lipid matrix, coated by the suspending agent PVA 9000 and the cationizing agent DEAE-dextran, as previously described [SLNescribed . BrieflyEE, Nile Red was dissolved in the dichloromethane of the organic phase, whereas in SLNHM Nile Red was dissolved in dichloromethane and added during the first 30 s of the sonication step. Finally, in the case of SLNC, a solution of dichloromethane containing Nile Red was added during the re-melting step.When required, Nile Red was incorporated in the preparation of all SLNs to label them. In the case of SLNIn order to prepare SLN-based vectors, an aqueous solution of protamine (P) was added to the nucleic acid, mRNA or pDNA. Then, a solution of polysaccharide, dextran (DX) or hyaluronic acid (HA) dissolved in water was added. Finally, the SLN suspension was added to the previous complexes and incubated for 20 min. Electrostatic interactions between negative and positive charges of the components led to the formation of the final vectors, in which complexes were adsorbed on the surface of the cationic SLNs.The weight ratios of the components of the formulations are summarized in w) was incorporated into the above mentioned vectors. The final concentration of PVA was 1% (w/v).In order to obtain viscous formulations suitable for ocular delivery to animal models, an aqueous solution of PVA  of 3 batches of SLNs and vectors were measured by Dynamic Light Scattering (DLS) and ζ-potential was determined by Laser Doppler Velocimetry (LDP). These measurements were carried out in a ZetaSizer Nano ZS  after the appropriate dilution of the samples in Milli-Q™ water .TM. The gel ran for 60 min at 75 V, then it was immediately analyzed with the Uvitec Uvidoc D-55-LCD-20 M Auto transilluminator . In order to evaluate binding capacity, vectors were diluted in MilliQTM water to a final concentration of 0.12 µg mRNA/µL in the gel. Protection from RNase I digestion was analyzed by the addition of 6 U RNase I/µg mRNA; mixtures were incubated at 37 °C for 40 min in a heater. Then, the samples were removed from the heater and mixed with an SDS solution , at room temperature. The same SDS solution was added to the vectors to unbind the mRNA in the release studies. Two controls for the integrity of the mRNA were included in the assay: RiboRuler High Range RNA Ladder and untreated CleanCapTM EGFP mRNA (5moU).Studies of mRNA binding capacity, protection from RNase I digestion and release from the vectors were performed in a 1.2% agarose gel electrophoresis labelled with Gel RedTM was used. The gel was analyzed with the Uvitec Uvidoc D-55-LCD-20 M Auto transilluminator  after running for 30 min at 120 V. The binding capacity was evaluated by adding vectors diluted in MilliQTM water to a final concentration of 0.03 µg pDNA/µL in the gel. For DNAse I protection, the same concentration was exposed to 1 U DNase I/2.5 μg pDNA and then incubated at 37 °C for 30 min in a heater. The samples were removed from the heater and mixed with an SDS solution (4%) to a final concentration of 1% at room temperature. The same SDS solution was added to the vectors to unbind the plasmid in the release studies. Two controls for the integrity of the pDNA were included in the gels: 1 kb pDNA ladder from NIPPON Genetics Europe  and untreated pcDNA3-EGFP plasmid.In order to study the pDNA binding capacity, protection against DNase I digestion and release from the vectors, 0.7% agarose gel electrophoresis with Gel RedCrison Basic 20 pH meter  was employed to determine the pH of the vectors. Measures were carried out in triplicate and the pH meter was calibrated daily.in vitro studies. HCE-2 cell line was maintained in a DMEM/F-12 GlutaMAXTM medium, which was supplemented with 15% (v/v) heat-inactivated fetal bovine serum (FBS), insulin (4 mg/mL), Epidermal Growth Factor (EGF) (10 ng/mL), and Penicillin–Streptomycin (1% v/v) and incubated at 37 °C with 5% CO2. Cells were sub-cultured once a week using Trypsin/EDTA in flasks earlier treated with Attachment Factor. The medium was renewed every 2 days. Cells from passages 1 to 4 were used to perform all in vitro assays.Human corneal epithelial (HCE-2) cell line was used in order to perform mRNA-based vectors were prepared 72 h before their addition and maintained at 4 °C before their use.2. The medium containing the vectors was removed after the incubation time, and the cells were refreshed with 1 mL of the complete medium. The cells were kept growing during 48 h or 72 h in the case of mRNA- or pDNA-vectors, respectively.After incubation with Attachment Factor, the cells were cultured on 24-well plates for 72 h at a density of 70,000 cells/well, until the cells were adhered and were able to create a monolayer. Later, part of the medium was removed, leaving enough volume to cover the cells, and a total volume of 75 µL of each vector diluted in Hank’s Balanced Salt solution (HBS)  was added to each well for 4 h in the incubator at 37 °C in a 5% COThe percentage of transfected cells and intensity of fluorescence, indicative of the amount of GFP produced, as well as cell viability, were measured using a CytoFLEX flow cytometer (Beckman Coulter). For this purpose, the cells were washed with 500 µL of PBS and then detached by incubation with 300 µL of Trypsin/EDTA for 5 min. After the centrifugation of cell suspension at 1000 rpm for 5 min, the supernatant was removed, and the pellet of cells was resuspended in 500 µL of PBS. Ten thousand events were collected for each sample. Transfection efficacy was measured at 525 nm (FITC) , and celThe effect of temperature on cell transfection was studied by the incubation of HCE-2 cells at 4 °C for 30 min, prior to the addition of the vectors. The cells were maintained for 4 h at 4 °C, and then the cells were treated as explained above.2. Once the incubation time had finished, the culture medium was removed and the cells were detached from the plates, as described in The internalization of the vectors by the HCE-2 cells was studied by using vectors containing SLNs labelled with the fluorescent Nile Red dye (λ = 590 nm), as previously described  and as eAdditionally, the effect of temperature on cellular uptake was studied by the incubation of cells at 4 °C for 30 min before the incorporation of the vectors. Once the Nile Red-labelled vectors were added to the cell cultures, the cells were maintained at 4 °C for an additional 2 h. Finally, the cells were collected to evaluate the vector uptake by flow cytometry, as described above. In this case, the percentage of positive cells corresponds to the cells that have uptaken vectors labelled with Nile Red over the total cells.2 for 24 h. Then, they were treated with 75 µL of vector equivalent to 0.8 µg of CleanCapTM Cyanine 5 EGFP mRNA (5moU) as nucleic acid. After 4 h, the slides were washed with PBS, fixed with PFA 4% and covered with the mounting fluid DAPI-fluoromount-G™, used to label the nuclei. Then, a Leica DM IL LED Fluo inverted microscope  was used to analyze the slides.A density of 150,000 cells in 1 mL per well was seeded in Millicell EZ slides (Millipore) and incubated at 37 °C and 5% COg for 2 min. For the intracellular IL-10, the cells were washed with 300 μL of PBS twice, and then 400 μL of reporter lysis buffer 1× was added. Finally, the plate was frozen to complete the lysis of cell culture. After thawing, each well was detached by a scrapper and the lysate was centrifuged at 12,000× g for 2 min at 4 °C. A total of 100 μL of each sample was added to a 96-well plate that was covered with the corresponding capture antibody; then the assay was performed according to the manufacturer’s instructions.To measure the levels of IL-10 expressed by the cells aft er the addition of the complexes, an Enzyme-linked Immunosorbent Assay (ELISA) kit was carried out. The secreted and intracellular IL-10 were quantified 48 h and 72 h after the addition of the mRNA- and pDNA-bearing vectors encoding human IL-10, respectively. For the secreted IL-10, the medium of each well was removed and centrifuged at 12,000× in vivo studies.Five-week-old male BALB/cOlaHsd mice, with a weight ranging between 20 and 25 g (Envigo), were employed for the ad libitum access.The use of the mice (license M20/2018/142) was approved by The Animal Experimentation Ethics Committee of the University of the Basque Country UPV/EHU following the Spanish and European Union (EU) laws. All the procedures were followed in accordance. The animals were accommodated under controlled temperature, humidity, and 12 h day-night cycles, with food and water The mice were anesthetized with 1–2% isoflurane  in air, at a flow rate of 0.5–1 L/min with the aim of preventing distress during experimental manipulation.® O.C.T.TM and shaken at room temperature for 2 h. Finally, the eyeballs were stored in 100% Tissue-Tek® O.C.T.TM to freeze at 80 °C for future studies.The mice were humanely euthanatized by cervical dislocation, and then their eyes were removed. After the enucleation, the eyes were washed in a physiological saline solution, fixed with 4% PFA during 30 min and washed with PBS for 5 min. Then, the eyes were immersed in 30% sucrose in PBS at 4 °C until the eyes precipitated. Then, half of the volume was removed and substituted with Tissue-Tekw), and administered to the mice by eye drop instillation. The administration of the nanosystems was carried out in 2 doses over 3 days. In each dose, 3 instillations of 2.5 µL at 3 min intervals were carried out, administering a final dose of 4.5 µg of nucleic acid per day.The formulations described in EE formulation, where the mice were sacrificed both at 24 h and 48 h. For IL-10 expression studies, the mice were sacrificed at 24 h. Then, the eyeballs were extracted, fixed and histologically evaluated by sections of 14 µm on a cryostat .The mice were sacrificed 24 h or 48 h after the last dose depending on the formulation administered. For GFP expression studies, the mice were sacrificed at 48 h, except in the case of mRNA-HA-SLNIn order to evaluate the gene expression, two different transfection studies were carried out. In the first one, vectors contained the nucleic acid that encodes GFP, whereas in the second one vectors were bearing the nucleic acid that encodes IL-10. Both transfections were evaluated qualitatively by immunofluorescence. Sections were washed with a PB buffer. The samples were blocked and permeabilized employing a solution of 20% PB, 0.3% Triton X-100, 10% goat serum, and water q.s. 100%. Then, the respective primary antibody, anti-GFP or anti-IL-10, was added and incubated for 24 h at 4 °C. Secondary antibody goat anti-rabbit IgG Alexa Fluor 488 was added in both transfection assays after washing for 30 min protected from light. Finally, after washing and drying the samples, they were mounted with DAPI-Fluoromount-G. Tissue sections were examined by a Zeiss LSM800 confocal microscope . The overlapping of fluorescence emission spectra was avoided by sequential acquisition. Six sections for each cornea were analyzed as representations of the entire tissue.p < 0.05 was considered statistically significant. Data are shown as mean ± standard deviation (SD).IBM SPSS Statistics 26 (IBM) software was used to perform the statistical analysis, and the Saphiro–Wilk test and Levene test were employed for the evaluation of homogeneity and variance, and normal distribution of samples, respectively. Student’s t-test was used to compare means from two independent groups and ANOVA for multiple comparisons, followed by Bonferroni or T3 Dunnet post-hoc, depending on the results of the Levene test of homogeneity of variances. p < 0.001) were observed in terms of particle size and superficial charge among all SLNs. SLNC showed the highest particle size and the lowest superficial charge, whereas SLNHM showed the smallest size and the highest superficial charge. The Particle Size Distribution Intensity Diagram of SLNHM has been included as an example in (ample in .TM EGFP mRNA (5moU) or IL-10 mRNA customized by TriLink BioTechnologies. All formulations were prepared with protamine (P). Moreover, an aqueous solution of polysaccharide, either dextran (DX) or hyaluronic acid (HA), was incorporated into the SLNEE and SLNHM vectors. Since SLNC vectors contain DEAE-dextran in their composition, only HA was used to prepare the mRNA-HA-SLNC vector. The particle size of vectors containing EGFP mRNA ranged from 132.3 to 348.4 nm, PDIs were lower than 0.4, and the surface charge ranged from +9.5 to +43.9 mV. A PDI value less than 0.4 is related to the homogeneity in the size of the particles of the sample and it demonstrates a monodisperse sample population, which is considered acceptable for drug delivery [p < 0.001) in terms of size were observed in both mRNA-SLNHM formulations and mRNA-HA-SLNC with respect to the rest of the formulations. Regarding the superficial charge, significant differences (p < 0.001) were observed between SLNC vectors , as well as mRNA-DX-SLNEE , with respect to the rest of the formulations. Particle Size Distribution Intensity Diagram of mRNA-HA-SLNHM bearing CleanCapTM EGFP mRNA (5moU) has been included as an example in (delivery . Signifiample in .p < 0.001) were observed between the vectors prepared with different SLNs with respect to the rest of the formulations in terms of particle size. Regarding the superficial charge, significant differences (p < 0.001) were observed between mRNA-SLNC and mRNA-HA-SLNC with respect to the others. IL-10 mRNA vectors showed a smaller particle size than those prepared with GFP mRNA, ranging from 116.9 to 283.9 nm, with a PDI lower than 0.3 and a superficial charge from +19.4 to +49.2 mV. Significant differences  were noticed with regard to particle size and ζ-potential between formulations. In the case of pUNO1-hIL10 vectors, the particle size ranged from 101.1 to 193.7 nm, PDIs were less than 0.3 except for pDNA-HA-SLNHM, which was 0.48. The superficial charge ranged from +41.4 to +44.7 mV. Significant differences (p < 0.001) were reported in terms of size between formulations.No significant differences in particle size, PDI, or ζ-potential was observed when SLNs were labelled with Nile Red (data not shown).HM and SLNC vectors to bind, protect, and release mRNA  or plasmid pcDNA3-EGFP combined with PVA were topically administered as eye drops to mice in order to assess the capacity of transfection in the corneal epithelium. Vectors bearing CleanCapGFP was detected in 100% of the sections analyzed. All formulations were able to transfect and produce GFP in the corneal epithelium. GFP produced by naked mRNA was difficult to observe, whereas the intensity of fluorescence of GFP was higher when mRNA was formulated in the vectors.EE and mRNA-DX-SLNHM was localized continuously along the epithelium surface. In the case of mRNA-HA-SLNEE, mRNA-HA-SLNHM, and mRNA-SLNC, uninterrupted segments of GFP were observed. In contrast, GFP in the corneas transfected with mRNA-HA-SLNC was localized discontinuously.GFP produced by mRNA-DX-SLNHM than with pDNA-DX- SLNHM. pDNA-based formulations were used as controls for the mRNA-based ones; naked pDNA (GFP and IL-10) was previously evaluated [Regarding transfection of the corneas with the nanocarriers prepared with pDNA, GFP was detected in a wider area with pDNA-HA-SLNvaluated .EE) after 24 h, it was observed that GFP was detected in a larger surface area and with a higher intensity of fluorescence than after 48 h .IL-10 was observed continuously along the corneal epithelium in all analyzed sections. The intensity of the fluorescence signal was higher when the corneas were treated with the nanosystems than in the case of the naked mRNA. Slight differences were found among the different vectors, although with mRNA-DX-SLNEE, prepared by evaporation/emulsification, SLNHM obtained by hot-melt emulsification, and SLNC prepared by coacervation. The development of new nanomedicine products for mRNA-based therapies has emerged as an attractive and promising tool in the field of advanced therapies. In this study, we have developed nanovectors based on SLNs for nucleic acid delivery. Three different methods were used to prepare the SLNs: SLNEE and SLNHM, made up with the same chemical components but prepared by different methods, varied in particle size. Indeed, it ranged from 90 nm in the case of the hot emulsification procedure, which avoids the use of organic solvents, but involves a high operating temperature, to 200 nm in the case of the evaporation/solvent method. The solvent-free coacervation method, which exploits different compositions compared to the previously mentioned techniques, led to the highest particle size.The preparation method influenced the physicochemical features of the SLNs, in terms of particle size and surface charge. In particular, SLNThe final vectors were made up by electrostatic interactions among SLN, nucleic acid (mRNA or pDNA), and different ligands. Differences regarding the physicochemical characteristics of the SLN may lead to variations in the arrangement of the remaining components, since they are adsorbed on the SLN’s surface to form the final vector. Therefore, the transfection capacity can be influenced. Firstly, the genetic material was condensed with P, which contributes to binding and protecting the nucleic acid extra and intracellularly due to its cationic character ,40. SecoFor corneal transfection, small-sized particles between 10 and 1000 nm reduce eye irritability after topical administrations. Moreover, they show mucoadhesive properties, which help to prolong the residence time and, consequently, to increase the drug bioavailability in the ocular tissues ,44. The EE and mRNA-SLNHM vectors, differences in the condensation degree were observed , those containing DX were more effective than those containing HA. The mRNA-vectors, including the SLNs prepared by coacervation, hardly produced IL-10. The levels of IL-10 secreted by cells treated with the vectors prepared with SLNHM and either mRNA or pDNA were similar (EE were higher; in particular, the most effective formulation, mRNA-DX-SLNEE, showed IL-10 levels almost three-folds higher. Transfection studies with the nanovectors bearing pDNA or mRNA encoding the anti-inflammatory cytokine IL-10 were also carried out in HCE-2 cells. The IL-10 was measured in the culture media and at the intracellular level. The mRNA vectors most efficient in terms of IL-10 production were those containing the SLNs prepared by the solvent/evaporation method. For the same kind of SLNs (SLN similar . It is ein vivo studies, the thickening agent PVA was added to the vectors. The non-ionic and synthetic biodegradable hydrophilic polymer PVA [The formulation of nanodelivery systems plays a crucial role in the development of medicinal products based on gene therapy, and specifically, in ocular gene therapy. An optimal ophthalmic drug formulation should comply to an adequate bioavailability, an increased permeability, an improved stability against degradation, a prolonged retention on the eye surface, and an augmented interaction with the cornea and targeted delivery . Indeed,ymer PVA  is approymer PVA . PVA hasymer PVA . These pymer PVA ,57. In eymer PVA . Ophthalymer PVA ,59. The ymer PVA . Our forIn vivo studies in mice were first carried out to evaluate the formulations containing mRNA or pDNA for GFP expression. Since GFP, once produced, remains at the intracellular level, it allowed us to identify the corneal layers where transfection occurs, after the instillation on the mice ocular surface with these vectors. Since the cornea is a complex structure, mRNA-delivery systems are engineered to induce the therapeutic protein expression in the cornea, specifically in the stratified and renewable epithelial layer, where a high number of cells can be transfected. Another alternative could be the transfection of the innermost layer of the cornea, the endothelial layer, associated with a difficult accessibility. Indeed, this layer contains a low number of cells that do not undergo division and gene expression could be maintained for longer times. To this aim, DNA could be most advantageous, since it provides a more persistent transgene production than mRNA. However, although we have previously shown the capacity of DNA-based formulations to transfect the cornea [e cornea , mRNA poe cornea , a shortin vivo and showed a higher intensity of fluorescence than naked mRNA. Thus, the SLNs resulted necessary to obtain a high transfection efficiency. These formulations were able to transfect only the epithelial cells but not the inner layers of the cornea, regardless of their different particle size. The intensity of fluorescence of GFP observed in the HCE-2 cells in vitro, representative of the protein production, was similar for all formulations, and it seems to correlate better with the in vivo results than with the percentage of transfected cells in vitro. Nevertheless, the lack of a strict correlation between in vitro and in vivo studies [All mRNA-based formulations included in this experimental work transfected the cornea  studies ,63,64,65EE vector, the best performing formulation in in vitro experiments in HCE-2 cells (C vectors also showed a high capacity to produce IL-10, despite the low efficacy observed in vitro. It should be considered that the transfection efficacy of the nanovectors could be increased in inflamed corneas, as the histological structure of corneal layers would be altered and disorganized, resulting in a potentially increased permeation of the nanosystems into the tissue. Within this concern, in previous studies with SLN-based nanosystem containing pDNA as the nucleic acid, vectors prepared with the polysaccharide HA showed the highest transfection efficacy [Finally, we evaluated the capacity of our formulations to induce the production of the anti-inflammatory cytokine IL-10 . In view-2 cells , the int-2 cells . mRNA-HAefficacy . pDNA anin vitro studies. We have demonstrated that the HCE-2 cells’ transfection is favoured by energy-dependent mechanisms, but the production of protein is more efficient when the vectors are taken up by energy-independent mechanisms. However, the lack of correlation observed between in vitro and in vivo assays has highlighted the necessity to perform in vivo studies at the earliest phases of the pharmaceutical development of nucleic acid delivery systems. In the present work, SLNs allowed the ability to obtain a high transfection efficiency in vivo. Therefore, topical administration to mice of eye drops containing mRNA formulated in SLNs has shown to be a feasible strategy to tackle corneal inflammation by de novo fast IL-10 production from corneal epithelial cells.The nature of the nucleic acid and the target cell are key points for the design and optimization of nanosystems aimed at ocular gene delivery, with mRNA being more sensitive to the formulation-related factors than pDNA. Nanomedicinal products design is based, initially, on


Although lower high-density lipoprotein cholesterol (HDL-C) levels are considered a risk factor for cardiovascular disease (CVD), experimental evidence suggest that aging, inflammation, and oxidative stress may remodel HDL-C, leading to dysfunctional HDL-C. Population studies on HDL-C and loss of the glomerular filtration rate (GFR) reported inconsistent results, but they used inaccurate estimates of the GFR and may have been confounded by comorbidity.n=1627) and at the follow-up (n=1324) after a median of 5.6 years. We also investigated any possible effect modification by low-grade inflammation, physical activity, and sex.We investigated the association of HDL-C levels with risk of GFR loss in a general population cohort; the participants were aged 50–62 years and did not have diabetes, CVD, or chronic kidney disease (CKD) at baseline. The GFR was measured using iohexol-clearance at baseline (2 per year) in multivariable adjusted linear mixed models and logistic regression . Effect modifications indicated a stronger association between high HDL-C and GFR loss in physically inactive persons, those with low-grade inflammation, and men.Higher HDL-C levels were associated with steeper GFR decline rates and increased risk of rapid GFR decline (>3 ml/min per 1.73 mHigher HDL-C levels were independently associated with accelerated GFR loss in a general middle-aged nondiabetic population.  This paradigm has been challenged, as clinical trials designed to increase HDL-C levels have failed to show any clinical benefits,Previous experimental studies and human data indicate that there is a complex association between higher HDL-C levels and vascular dysfunction, atherosclerosis, and kidney dysfunction.19Although several studies have reported an association of low HDL-C levels with incident CKD, both low and high HDL-C levels were associated with a GFR loss, CKD progression and end-stage kidney disease in a study of nearly 2 million male US veterans.23,,,In this study, we investigated the association of HDL-C levels with decline in measured GFR in persons from the general population without pre-existing CKD, diabetes, or CVD. Because physical activity, low-grade inflammation, and sex have been shown to influence HDL-C functionality,The Renal Iohexol Clearance Survey (RENIS) is a substudy of the sixth wave of the population-based Tromsø Study (Tromsø 6), Northern Norway. A 40% random sample of individuals in the municipality of Tromsø aged 50–59 years and all individuals aged 60–62 years  were invited, and 3564 (65%) individuals completed the main study. Participants who did not report having a history of myocardial infarction, angina pectoris, stroke, diabetes, or kidney disease were invited to join RENIS-T6 . A totalThe RENIS study was approved by the local ethics committees and performed in accordance with the guidelines of the Declaration of Helsinki. All subjects provided written informed consent.am and 10:00 am to have their GFR measured and blood samples drawn. Participants with symptoms of intercurrent illness had to reschedule their appointments.The RENIS-T6 and RENIS-FU were conducted at the Clinical Research Unit at the University Hospital of North Norway. The participants fasted at home from midnight and were asked to drink 2 glasses of water in the morning before they came to the hospital between 8:00 ,The GFR was measured at baseline and at follow-up with single-sample plasma clearance of iohexol (mGFR) as previously described in detail.,The fasting serum glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol, and HDL-C concentrations were measured by a Modular P800  instrument. The HDL-C level was categorized as low when it was ≤1.0 mmol/l (≤40 mg/dl), intermediate when it was 1.1–1.6 mmol/l (41–61 mg/dl), and high when it was >1.6 mmol/l (>61 mg/dl), as suggested previously.Serum creatinine analyses were performed using a standardized enzymatic assay, and cystatin C was measured by particle-enhanced turbidimetric immunoassay.Three samples of first-void morning spot urine were collected on consecutive days prior to the GFR measurements. The urinary albumin and creatinine concentrations were measured in fresh urine, as previously reported.1c were measured in the main Tromsø 6 study, as described previously.,High-sensitivity C-reactive protein (Hs-CRP) and HbABlood pressure was measured 3 times in a seated position after a 2-minute rest period. The average of the second and third measurements was used in the analyses.,o2max) was moderate in a study where questions were repeated and physical fitness was assessed by the measurement Vo2max .A health questionnaire included questions on tobacco and alcohol use, current medications, and physical activity related to the frequency and intensity of leisure-time physical exercise.We dichotomized physical activity, as reported in a previous publication, as follows: active  or inactive .Alcohol use was categorized according to the frequency at which subjects drank alcohol . Individuals were categorized as being a daily smoker, previously being a daily smoker, or never being a daily smoker.,n=88) in the RENIS-FU, this method allowed us to estimate the 3 variance components in the unstructured covariance matrix of the model. The association of the HDL-C level with the rate of GFR decline was analyzed by including 2-way interaction terms between the HDL-C variable and the time variable.A linear trend across groups by HDL-C levels was tested with linear or median regression for continuous variables and with logistic regression for dichotomous variables. The association between the baseline HDL-C levels  and change in the GFR was analyzed by a linear mixed regression model with a random intercept and slope. All 1627 participants with 1 to 3 GFR measurements were included in the analyses because linear mixed models allow for missing observations at 1 or more time points as long as the observations are missing at random.n = 1324). Rapid GFR decline was defined as a rate of GFR decline steeper than 3 ml/min per 1.73 m2 per year , a cut-off that has been used in previous studies.,,The association of HDL-C with the odds of rapid GFR decline was analyzed using logistic regression for those with at least 1 follow-up .n = 826) were women, the mean (SD) age was 58.1 (3.8) years, and the mean GFR was 93.9 (14.4) ml/min per 1.73 m2. The median HDL-C level was 1.5 (interquartile range 1.2–1.8) mmol/l (58 [interquartile range 46–70] mg/dl). The distribution of HDL-C levels at baseline is shown in The study population characteristics at baseline grouped by low, intermediate, and high levels of HDL-C are shown in 2 per year. A rapid GFR decline (GFR loss > 3.0 ml/min per 1.73 m2 per year) was observed in 70 men and 68 women. Higher HDL-C levels were associated with a steeper annual GFR decline and an increased odds ratio (OR) of rapid GFR decline  and OR 2.7  per doubling (log2) in HDL-C) (P = 0.01) for rapid GFR decline, compared to subjects with HDL-C ≤1.0 mmol/l. There was no relevant collinearity between covariates (mean and maximum variation inflation factor was 1.3 and 2.2), and the logistic regression models were well calibrated according to the Hosmer-Lemeshow statistics.The unadjusted mean rate of GFR decline was –0.84  ml/min per 1.73 mn HDL-C)  and 3. SP value for interaction < 0.01 and 0.04); the results stratified by physical activity are shown in follow-up – GFRbaseline / observation time) were also calculated using linear regression; the results were essentially the same as in the mixed model analyses and are shown by physical activity group in The associations with HDL-C were modified by physical activity for both the mean GFR decline rate (mixed linear regression) and odds ratio for rapid decline    and S4.2 at follow-up. The OR per doubling of HDL-C for incident CKD was 4.6  in the fully adjusted model  and 38 persons with incident diabetes (fasting glucose level of >7.0 mmol/l or an HbA1c level of ≥6.5%) at follow-up. The results remained almost identical (To test whether a phase of hyperfiltration (increasing GFR from baseline to follow-up) in subjects with low HDL-C could have influenced our results, we excluded 138 persons with incident prediabetes  as a dependent variable. The HDL-C levels were not associated with the mean GFR decline or risk of rapid eGFR decline using eGFRcys, but a similar tendency to the results using the measured GFR was found for eGFRcrea and eGFRcreacys, including a significant interaction between a rapid decline and physical activity using eGFRcreacys .In middle-aged subjects from the general population without pre-existing diabetes, CVD, or CKD, we found that higher HDL-C levels were independently associated with a steeper GFR decline and an increased risk of rapid GFR decline during a median of 5.6 years of follow-up.,,46Previous epidemiologic studies of HDL-C and the risk of kidney disease, including 3 Mendelian Randomization studies, reported inconsistent results.8,All these studies used estimates of GFR, and some studies included persons with diabetes or CKD. In most studies, the populations were not representative of the general population, and in several studies they did not adjust for relevant confounders.In the largest study of HDL-C levels and renal outcomes, consisting of 1,943,682 male veterans, the authors reported a U-shaped association of HDL-C with eGFR decline and end-stage kidney disease.HDL-C is traditionally regarded as “good” cholesterol, and the association of higher HDL-C with the loss of the GFR may seem counterintuitive. Several hypotheses can be raised as explanations for our findings.,Persons with high HDL-C levels may suffer from other conditions that can influence the GFR decline rate, such as inflammation or alcohol abuse.,,Experimental evidence suggests that high levels of HDL-C per se, or higher levels of dysfunctional HDL-C, contribute to endothelial dysfunction and vascular disease.We did not measure HDL-C dysfunction in the current study; however, previous studies have shown that HDL-C from healthy nonobese elderly persons contains higher levels of glycosylated apoA-1 and exhibits a lower antioxidative ability than does HDL-C from younger persons.Smaller modified HDL-C particles and HDL components, such as ApoA1, may interact with several renal cell classes, as they are filtered in the glomeruli and reabsorbed in the proximal tubuli.,Associations of higher HDL-C levels with the GFR change rate and risk of rapid GFR decline were found in subjects who reported performing little or no physical activity, and the association with the GFR change rate was significant for men and subjects with higher hs-CRP levels only. Although the results of subgroup analyses should be interpreted with caution, we speculate that the effect modifications of hs-CRP, physical activity, and sex may be explained by altered HDL-C functionality. Experimental studies showed that low-grade inflammation modulates the composition and function of human HDL-C, leading to the loss of endothelial protective properties.,,Physical activity, on the other hand, and particularly aerobic exercise, has been shown to reduce low-grade inflammation and to improve the antioxidant and anti-inflammatory effects of HDL-C.We observed a stronger association of HDL-C with the mean GFR decline rate in men than in women. Several sex-specific differences have been reported in the etiology and epidemiology of CKD, but the underlying mechanisms are unclear. Sex differences in vascular function, HDL oxidation leading to dysfunctional HDL-C, and inflammation in the kidneys may potentially influence the association of HDL-C with GFR decline.The main strength of the current study is the GFR measurements in a well-described general population cohort. Our results were robust when both linear mixed and logistic regression models were used. Some limitations should be mentioned. We investigated middle-aged persons mainly of North European ancestry; thus, our results cannot necessarily be generalized to other age groups or ethnicities. We did not include markers of dysfunctional HDL-C or objective measures of physical activity. The observation time was limited to 5.6 years, and the vast majority of subjects had 1 follow-up GFR only.We conclude that higher HDL-C levels were associated with a steeper GFR decline rate and increased the risk of rapid GFR decline in middle-aged subjects without diabetes and pre-existing CKD. Effect modifications indicated stronger associations of HDL-C with GFR loss in physically inactive persons, in those with higher hs-CRP levels and in men. This complex association of HDL-C with GFR loss should be addressed in future studies.BOE, TM, and VTS report an unrestricted grant from Boehringer-Ingelheim to the RENIS-FU study. The funding source had no role in the design and conduct of the study. ITE, JVN, MDS, RR, and TJ have no relevant financial relationships to disclose.


Theta oscillations over the posterior medial frontal cortex (pMFC) and lateral prefrontal cortex (LPFC) play vital roles in sustained attention. Specifically, pMFC power and pMFC-LPFC synchronization correlate with cognitive control in sustained-attention-related tasks, but the causal relationships remain unknown. In the present study, we first analyzed the correlation between EEG theta oscillations (characterized by time-frequency power and phase-based connectivity) and the level of sustained attention (Experiment 1) and then utilized transcranial alternating current stimulation (tACS) to modulate theta oscillations and in turn observed its effects on sustained attention (Experiment 2). In Experiment 1, two time-frequency regions of interest (ROIs) were determined, in which high/low time-frequency power and high/low phase-based connectivity corresponded to high/low-level sustained attention. In Experiment 2, time-frequency power and phase-based connectivity of theta oscillations were compared between the sham and tACS groups within the time-frequency ROIs determined in Experiment 1. Results showed that phase-based connectivity between pMFC and LPFC significantly decreased in the tACS group compared with the sham group during the first five minutes of the poststimulation period. Moreover, a marginal trend existed that sustained attention was downregulated by tACS in the same time interval, suggesting that theta phase synchronization between pMFC and LPFC may play a causal role in sustained attention. Sustained attention could be defined as “the ability to self-sustain mindful, conscious processing of stimuli whose repetitive, non-arousing qualities would otherwise lead to habituation and distraction to other stimuli” . AlthougA large number of studies have suggested that sustained attention correlates closely with cortical oscillations, extracted often from electroencephalography (EEG) signals. Integrated into the model proposed by Clayton et al., frontomedial theta (fm-theta) oscillations and the coordination between the posterior medial frontal cortex (pMFC) and the lateral prefrontal cortex (LPFC) play vital roles in sustained attention . SpecifiTranscranial alternating current stimulation (tACS), a noninvasive brain stimulation technique, is capable of providing causal evidence concerning the role of cortical oscillations in cognition , 11. By The present study is aimed at investigating the causal roles of theta-band pMFC-LPFC oscillations in sustained attention via tACS. To this end, we conducted two close-knit experiments in two steps. In Experiment 1, we used EEG to explore whether and how theta-band pMFC-LPFC oscillations were correlated with sustained attention. Two types of EEG features—time-frequency power on pMFC and phase-based connectivity between pMFC and LPFC—were analyzed. Based on the correlative evidence between these two features and the sustained attention obtained from Experiment 1, we further conducted Experiment 2 by applying theta-tACS (6 Hz) to specifically modulate the power as well as phase synchronization between pMFC and LPFC to affect the behavioral performance of sustained attention. Experiment 2 was expected to provide causal evidence between theta-band pMFC-LPFC oscillations and sustained attention, which would be fundamentally helpful in advancing our understanding of the neural mechanisms of sustained attention.A total of 10 volunteers  participated in this study. All participants were on-campus students and had a normal or corrected-to-normal vision. None of them suffered from any neurological or psychological disorder or took medication that interfered with the study. Before formal participation, they were required to rest well and to refrain from consuming caffeine or alcohol. Participants provided informed consent before the start of the experiment. The experiment was approved by the local ethics committee at Tianjin University.http://psychtoolbox.org) for MATLAB .The psychomotor vigilance task (PVT) is a simple but mentally demanding reaction time test, which is often used in research on sustained attention and fatigue . In the Ω during the whole experiment. After recording, raw EEG signals were cleaned from clear artifacts by visual inspection, filtered by high-pass filter with 0.1 Hz using EEGLAB software ), where the baseline was the average power at each frequency band from −400 ms to −200 ms before the onset of the stimulus and the time range from -200 ms to 800 ms was presented. To find significant different regions in the whole time-frequency domain, a cluster-based permutation test was used . Power dPhase-based connectivity is also known as phase synchronization or phase coherence . Here, wN is the number of trials and ℑ{} denotes the imaginary part of the cross-spectrum X between two electrodes. Compared with other indexes, such as phase-locking value (PLV), dWPLI is more insensitive to volume conduction and more appropriate for exploratory data-driven studies. Note that the spatial filter of the surface Laplacian [in which aplacian , also knaplacian , 26.t-test was utilized to compare the averaged dWPLI within that selected window.The Fz and F3 electrodes were used to measure activities over pMFC and LPFC, respectively, consistent with prior studies –30. ThenFifteen volunteers  who had a normal or corrected-to-normal vision and were also naïve concerning the stimulation participated in this study. Every participant took part in two experimental sessions: sham stimulation and tACS. The periods between the two sessions ranged from 1 day to 1 week. The sequence of applying two sessions was counterbalanced across subjects. All subjects provided informed consent before the start of the experiment. Influences of circadian rhythms were minimized by scheduling the sessions for all participants only in the afternoon. Three participants were excluded due to insufficient EEG data after preprocessing, leaving twelve participants  for analysis. The experiment was approved by the local ethics committee at Tianjin University.http://psychtoolbox.org) for MATLAB .In Experiment 2, we used a revised version of the psychomotor vigilance test . Particiπ circular area for each one. The electrodes named PISTIM, produced by Neuroelectrics, Spain, can work for both stimulation and EEG monitoring. Based on the fact that sustained attention was highly correlated with theta-band oscillations in Experiment 1, a sinusoidally alternating current of 1 mA (peak-to-peak) was applied at 6 Hz continuously lasting for 15 minutes. Six Hz was selected here as the center frequency in two interested frequency bands, 4-6 Hz and 5-8 Hz, obtained from Experiment 1. The stimulating current was ramped up over 20 seconds to 1 mA in both sham stimulation and tACS groups; however, the current was then faded into 0 mA in 20 seconds in the sham group. Participants reported that neither painful skin sensations nor phosphenes were induced, and particularly, they could not distinguish between the sham and tACS groups. Specifically, the phase difference between the two stimulating electrodes was set at 180° determined by the stimulation montage we used in this experiment.tACS was delivered by a battery-operated stimulator . The stimulator was connected to two Ag/AgCl electrodes with a 1 cm radius, which thus provided r of 0.10-0.30 is small, 0.30-0.50 is medium, and bigger than 0.50 is large [For every participant under each stimulation (sham stimulation or tACS), the behavior baseline was defined by the sum of the mean and the standard deviation of reaction times in the first 5 minutes. To rule out the possible confounding effect of individual baseline differences, we chose not to compare RTs directly, but the proportions of negative responses as follows. First, we specified six time intervals, namely, 0-5 min, 5-10 min,…, 20-25 min, and 25-30 min, respectively. Next, within each time interval, the number of sad expressions was divided by the number of all expressions. Last, these ratios of sad expressions  in six time intervals  under two conditions  were compared using two-way repeated measures ANOVA. Furthermore, to investigate the immediate effects after stimulation, considering the nonuniformity of behavioral data in the current dataset, the Wilcoxon signed-rank test on paired samples was performed to compare sustained attention in the fifth time interval (20-25 min) between the sham and tACS groups. To justify the sample size involved in Experiment 2, we report effect size estimates used with the Wilcoxon signed-rank test where an effect size is large . Also, wis large  and repois large .Because EEG signals during the stimulation (5-20 min) were introduced large amounts of artifacts, so we only analyzed the signals right before the stimulation (0-5 min) and right after the stimulation (20-30 min). Artifacts were cleaned by first visual inspection and then ICA. On average, 20% of the trials were rejected. As a consequence, for every participant, approximately 40 trials remained in the first 5 minutes, and approximately 80 trials remained in the last 10 minutes. We used those 40 trials in the first 5 minutes to represent the state of sustained attention before stimulation, and those 80 trials in the last 10 minutes to represent the state of sustained attention after stimulation.The time-frequency decomposition is the same as that in Experiment 1. The interested time-frequency windows determined in Experiment 1 were utilized to examine the difference between the sham and tACS groups. Within those windows, the averaged time-frequency power change at electrode Fz and the averaged phase-based connectivity change at Fz-F3 in the last two time intervals , relative to the baseline (0-5 min), under two stimulations  were compared using two-way repeated measures ANOVA. The relative change was used here due to different baseline states . Note thFurthermore, to investigate the immediate effects after stimulation, considering the nonuniformity of time-frequency data, the Wilcoxon signed-rank test on paired samples was performed to compare power and phase synchronization in the fifth time interval (20-25 min) between the sham and tACS groups, a similar way conducted in the previous tACS study .t (9) = −30.42, p < 0.0001, corresponding to Cohen's d effect size of d = 9.62 (a very large effect). These results indicated that high-level and low-level sustained attention can be well distinguished by their RTs in Experiment 1 .riment 1 . A similt (9) = 3.30, p < 0.01 (d effect size of d = 1.04 (a large effect). A similar conclusion was also reached by Bayesian t-tests, which showed moderate evidence against the null hypothesis that there was no power difference in the specified time-frequency window between high-level and low-level sustained attention (BF = 6.62).Time-frequency power on the pMFC (electrode Fz) was averaged across all subjects under high-level and low-level sustained attention, respectively Figures . Time-frp < 0.01 , correspt (9) = 2.68, p < 0.05 (d effect size of d = 0.846 (a large effect). A similar conclusion was reached by Bayesian t-tests, which showed nearly moderate evidence against the null hypothesis that there was no dWPLI difference in the specified time-frequency window between high-level and low-level sustained attention (BF = 2.97).DWPLI between pMFC (Fz) and LPFC (F3) was averaged across all subjects under high-level and low-level sustained attention, respectively Figures .  Figurep < 0.05 , correspTaken together, two different time-frequency windows were specified in which high-level sustained attention had both larger power and larger phase-based connectivity than low-level sustained attention in Experiment 1.F  = 0.448, p = 0.81, partial η2 = 0.01. The main effect of stimulation was nonsignificant, F  = 0.730, p = 0.411, partial η2 = 0.012. The main effect of time intervals yielded an F ratio of F  = 12.402, p < 0.001, indicating that the levels of sustained attention varied significantly across time intervals and sustained attention declined with the experimental time going on . These results indicated that tACS probably reduced sustained attention when the tACS was terminated (see A two-way repeated measures ANOVA was run to determine the effect of different stimulations (sham/tACS) over time intervals  on sustained attention, represented by the proportions of negative responses. The interaction effect between stimulation and time intervals on sustained attention was not statistically significant, g on see . To specated see . On the F  = 0.005, p = 0.947, partial η2 = 0.00004. The main effect of stimulation was nonsignificant, F  = 0.031, p = 0.864, partial η2 = 0.001. The main effect of time intervals was nonsignificant, F  = 1.158, p = 0.305, partial η2 = 0.007. To specifically see the immediate effect just after different stimulations over the fifth time interval  on sustained attention, a two-tailed Wilcoxon signed-rank test on paired samples was conducted and the results showed that the median relative power change of the tACS group had no significant difference with that of the sham group, p = 1, corresponding to an effect size r = 0. These results indicated that tACS did not modulate power in pMFC when the tACS was just terminated (A two-way repeated measures ANOVA was run to determine the effect of different stimulations (sham/tACS) over time intervals (20-25 min and 25-30 min) on the averaged power change relative to baseline (0-5 min) within the time-frequency window  determined in Experiment 1 . The intrminated . On the F  = 1.837, p = 0.203, partial η2 = 0.059. The main effect of stimulation was nonsignificant, F  = 0.007, p = 0.924, partial η2 = 0.00013. The main effect of time was nonsignificant, F  = 0.108, p = 0.749, partial η2 = 0.002. To specifically see the immediate effect just after different stimulations over the fifth time interval  on sustained attention, a right-tailed Wilcoxon signed-rank test on paired samples was conducted and the results showed that the relative dWPLI change of the sham condition was significantly larger than that of the tACS group, p = 0.026, corresponding to an effect size r = 0.566 (a medium effect). These results indicated that tACS reduced the phase-based connectivity between pMFC and LPFC when the tACS was just terminated (A two-way repeated measures ANOVA was run to determine the effect of different stimulations (sham/tACS) over time intervals (20-25 min and 25-30 min) on the averaged dWPLI changes relative to the baseline (0-5 min) within the time-frequency window  determined in Experiment 1 . The intrminated . A similTo investigate pMFC-LPFC oscillations in the process of sustained attention, we first analyzed the correlation between theta characteristics (time-frequency power and phase-based connectivity) and the level of sustained attention and then utilized tACS to modulate theta characteristics and in turn observed its effects on sustained attention. In Experiment 1, two interested time-frequency windows were determined, in which high/low time-frequency power and high/low phase-based connectivity (indexed by dWPLI) corresponded to high/low-level sustained attention. In Experiment 2, time-frequency power and phase-based connectivity were compared between sham stimulation and tACS groups within the time-frequency window determined in Experiment 1. There was no significant difference in time-frequency power between the two stimulation groups. However, phase-based connectivity between pMFC and LPFC significantly decreased in the tACS group compared with the sham group during the first five minutes of the poststimulation period. Moreover, there was a marginal trend that sustained attention was downregulated by tACS in the same time interval, suggesting that theta phase synchronization between pMFC and LPFC may play a causal role in sustained attention.In Experiment 1, the reaction time gradually increased as the experimental time went on, suggesting that the level of sustained attention gradually went down. This phenomenon, called vigilance decrement, was commonplace in PVT tasks . CorrespIn Experiment 2, there were no significant differences in time-frequency power and performance of sustained attention between the sham and tACS groups. However, trends were shown that in the first five minutes during the poststimulation period, namely, 20-25 min , sustained attention was probably downregulated by tACS. Most importantly, in the fifth time interval, phase-based connectivity between pMFC and LPFC significantly decreased in the tACS group compared with the sham group. These facts probably implied the causal relationship between sustained attention and theta phase synchronization between pMFC and LPFC. A large number of studies suggested that tACS with different phase differences can manipulate phase synchronization , 29, 30.There have been several similar studies in which theta band tACS over the pMFC was used to modulate sustained attention. For example, Rostami et al. aimed to modulate pMFC to influence sustained attention . They foThere are also some limitations to our study. The first limitation is our sample size across two experiments. We recruited 10 subjects in experiment 1 and 15 subjects  in Experiment 2. It is better to collect more data to get more robust results, especially for signal analysis in tACS. The second limitation of our study is the lack of a 0° tACS comparison set in Experiment 2. tACS with 180° phase difference used in our study mainly came from the bipolar setting of the stimulator. Next, high-density tACS could be used to realize 0° tACS –46, we cThe present study is aimed at investigating the causal roles of theta-band pMFC-LPFC oscillations in sustained attention via tACS. Results showed that in the first five minutes after tACS was terminated, theta-band phase-based connectivity between pMFC and LPFC significantly decreased; meanwhile, sustained attention had a declining trend. These findings implied the causal relationship between sustained attention and theta phase synchronization between pMFC and LPFC.